PROJECT SUMMARY The formation of a malignant tumor from a benign tumor has been strongly linked to the existence of heterogeneous cellular features within the tumor. Thus, elucidating the mechanisms underlying generation of tumor cell heterogeneity will be an important milestone to understand malignant tumor formation. We believe two applications of our work could be 1) earlier, more quantitative detection of malignancy based on molecular markers and 2) therapeutics that can freeze tumor development before metastasis or even revert back to a benign state. Our approach is to answer the following two questions: 1. What is the impact of the tumor-initiating cell?s identity on generating tumor cell heterogeneity? In experiments to date, the inability to avoid contamination of different cell origins, including tissue stem cells and non-stem cells into the resultant tumor mass has prevented identification of the precise molecular mechanisms. We address the contamination problem by establishing an experimental system that enables precise tracing of tumor cell lineage derived from single cells. Our method is the non-invasive, in vivo imaging of endogenous, single cell-derived tumor development in adult transparent zebrafish. This experimental system allows us to visualize individual tumor cells, and, more importantly, also selectively label and isolate the cells of interest for detailed cell characterization. Using this system, we have already imaged the tumors arising from single cells in the skin epithelium. Importantly, we have found some major differences in tumor cell proliferation between tumorous cell clusters and in tumor cell differentiation that lead to acquisition of invasive features. Our results identifying a subset of tumor cells displaying invasive features is nicely representing the generation of tumor cell heterogeneity within a tumor. We will perform long-term clonal analysis of tumor development and transcriptome analysis of isolated individual tumors to determine their cellular identity. Retrospective imaging analysis will be used to study the effect of a tumor?s microenvironment on generating tumor cell heterogeneity. 2. How is heterogeneity within a tumor established? Identification of the cell types of tumor-initiating cells will be followed by the investigation of the mechanisms driving a single tumor-initiating cell to become heterogeneous during multiple rounds of cell divisions. Our ability to distinctively label and isolate invasive tumor cells from non-invasive tumor cells enables us to investigate the gene expression modulations responsible for a subset of tumor cells acquiring invasive features during tumor development. We will further perform functional experiments in vivo to define the roles of specific molecular pathways in tumor invasion.